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Longsen Chang - One of the best experts on this subject based on the ideXlab platform.
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Blocking of negative charged carboxyl groups converts Naja atra neurotoxin to Cardiotoxin-like protein.
International journal of biological macromolecules, 2020Co-Authors: Yi-jun Shi, Jing-ting Chiou, Liang-jun Wang, Chia-hui Huang, Yuan-chin Lee, Ying-jung Chen, Longsen ChangAbstract:Naja atra cobrotoxin and Cardiotoxin 3 (CTX3) exhibit neurotoxicity and cytotoxicity, respectively. In the present study, we aimed to investigate whether the carboxyl groups of cobrotoxin play a role in structural constraints, thereby preventing cobrotoxin from exhibiting cytotoxic activity. Six of the seven carboxyl groups in cobrotoxin were conjugated with semicarbazide. Measurement of circular dichroism spectra and Trp fluorescence quenching showed that the gross conformation of semicarbazide-modified cobrotoxin (SEM-cobrotoxin) and cobrotoxin differed. In sharp contrast to cobrotoxin, SEM-cobrotoxin demonstrated membrane-damaging activity and cytotoxicity, which are feature more characteristic of CTX3. Furthermore, both SEM-cobrotoxin and CTX3 induced cell death through AMPK activation. Analyses of the interaction between polydiacetylene/lipid vesicles and fluorescence-labeled lipids revealed that SEM-cobrotoxin and cobrotoxin adopted different membrane-bound states. The structural characteristics of SEM-cobrotoxin were similar to those of CTX3, including trifluoroethanol (TFE)-induced structural transformation and membrane binding-induced conformational change. Conversely, cobrotoxin was insensitive to the TFE-induced effect. Collectively, the data of this study indicate that blocking negatively charged residues confers cobrotoxin with membrane-damaging activity and cytotoxicity. The findings also suggest that the structural constraints imposed by carboxyl groups control the functional properties of snake venom α-neurotoxins during the divergent evolution of snake venom neurotoxins and Cardiotoxins.
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dna aptamers against taiwan banded krait α bungarotoxin recognize taiwan cobra Cardiotoxins
Toxins, 2016Co-Authors: Ying-jung Chen, Chiayu Tsai, Longsen ChangAbstract:Bungarus multicinctus α-bungarotoxin (α-Bgt) and Naja atra Cardiotoxins (CTXs) share a common structural scaffold, and their tertiary structures adopt three-fingered loop motifs. Four DNA aptamers against α-Bgt have been reported previously. Given that the binding of aptamers with targeted proteins depends on structural complementarity, in this study, we investigated whether DNA aptamers against α-Bgt could also recognize CTXs. It was found that N. atra Cardiotoxin 3 (CTX3) reduced the electrophoretic mobility of aptamers against α-Bgt. Analysis of the changes in the fluorescence intensity of carboxyfluorescein-labeled aptamers upon binding toxin molecules revealed that CTX3 and α-Bgt could bind the tested aptamers. Moreover, the aptamers inhibited the membrane-damaging activity and cytotoxicity of CTX3. In addition to CTX3, other N. atra CTX isotoxins also bound to the aptamer against α-Bgt. Taken together, our data indicate that aptamers against α-Bgt show cross-reactivity with CTXs. The findings that aptamers against α-Bgt also suppress the biological activities of CTX3 highlight the potential utility of aptamers in regard to the broad inhibition of snake venom three-fingered proteins.
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Cardiotoxin iii suppresses the invasiveness of mda mb 231 cells by targeting proto oncogene tyrosine protein kinase src and reversing mesenchymal to epithelial transition
Toxicological & Environmental Chemistry, 2016Co-Authors: Peichien Tsai, Yiling Lin, Longsen Chang, Shinneren LinAbstract:ABSTRACTThe epithelial-to-mesenchymal transition is the first step required for breast cancer to initiate metastasis. The aberrant activation of proto-oncogene tyrosine-protein kinase Src regulates multiple functions during tumor progression. Cardiotoxin III, a basic polypeptide isolated from Naja naja atra venom, has been shown to exhibit anticancer activity; however, the effect of Cardiotoxin III on the epithelial-to-mesenchymal transition of cancer cells remains elusive. Exposure of MDA-MB-231 cells to Cardiotoxin III resulted in morphological changes and upregulation of E-cadherin with a concomitant decrease in N-cadherin and vimentin protein levels, resulting in the inhibition of cell migration and invasion. Cardiotoxin III induced downregulation of snail and slug expression. Simultaneously, Cardiotoxin III suppressed Src phosphorylation and downstream activation of focal adhesion kinase, of the docking protein p130cas, and of paxillin. In addition, Cardiotoxin III inhibited the phosphorylation of ex...
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involvement of both endoplasmic reticulum and mitochondria dependent pathways in Cardiotoxin iii induced apoptosis in hl 60 cells
Clinical and Experimental Pharmacology and Physiology, 2008Co-Authors: Chingming Chien, Longsen Chang, Shinneren Lin, Shenghuei YangAbstract:SUMMARY 1 Cardiotoxin (CTX) III, a basic polypeptide with 60 amino acid residues isolated from Naja naja atra venom, has been reported to have anticancer activity. In the present study, we investigated the mechanisms underlying the anticancer activity of CTX III in human leukaemia (HL-60 cells). 2 Cardiotoxin III activated the endoplasmic reticulum (ER) pathway of apoptosis in HL-60 cells, as indicated by increased levels of calcium and glucose-related protein 78 (Grp78), and triggered the subsequent activation of µ-calpain and caspase 12. 3 In addition, CTX III initiated the mitochondrial apoptotic pathway in HL-60 cells, as evidenced by an increased Bax/Bcl-2 ratio, the release of cytochrome c and activation of caspase 9. 4 In the presence of 50 µmol/L Z-ATAD-FMK (a caspase 12 inhibitor) and 100 µmol/L Z-LEHD-FMK (a caspase 9 inhibitor), the CTX III-mediated activation of caspase 9 and caspase 3 was significantly reduced. There was no significant effect of the caspase 12 inhibitor Z-ATAD-FMK on mitochondrial cytochrome c release. 5 Cardiotoxin III-mediated activation of caspase 12 was not abrogated in the presence of the caspase 9 inhibitor Z-LEHD-FMK, indicating that caspase 12 activation was not downstream of caspase 9. 6 These results indicate that CTX III induces cell apoptosis via both ER stress and a mitochondrial death pathway.
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Molecular Cloning and Evolution of the Genes Encoding the Precursors of Taiwan Cobra Cardiotoxin and Cardiotoxin-Like Basic Protein
Biochemical Genetics, 2004Co-Authors: Longsen Chang, Shu-kai Lin, Charling ChungAbstract:Genomic DNAs encoding the precursors of eight Cardiotoxins and two Cardiotoxin-like basic proteins (CLBP) were isolated from the liver of Naja naja atra (Taiwan cobra). The Cardiotoxin and CLBP genes have three exons like α-neurotoxin precursors. The promoter regions of these genes are highly conserved and contain the consensus transcriptional factor-binding sites for TBP, NF-1, CACCC-binding site, Spl and EFII, suggesting that these genes are regulated using similar transcriptional mechanisms. The introns and flanking regions of these genes share a high degree of nucleotide sequence identity, but except for the signal peptide domain the protein-coding regions are much more diversified than introns. The ratio of nonsynonymous to synonymous substitution is higher than one, reflecting that adaptive selection occurred during the evolution of Cardiotoxin and CLBP proteins. Phylogenetic trees separate CLBPs and Cardiotoxins into two clusters, suggesting that the CLBP gene and the Cardiotoxin gene diverged earlier before the appearance of numerous Cardiotoxins and CLBP.
Kandiah Jeyaseelan - One of the best experts on this subject based on the ideXlab platform.
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Cytotoxic potency of Cardiotoxin from Naja sputatrix: development of a new cytolytic assay.
Biochemical Journal, 2002Co-Authors: Arunmozhiarasi Armugam, Kandiah JeyaseelanAbstract:The possible involvement of specific regions/loops of Cardiotoxin from Naja sputatrix venom in mediating its cytolytic activity is evaluated using a new cytolytic assay. In this assay, the amount of chloramphenicol acetyltransferase (CAT) that is released upon lysis of the cellular membranes by the cytotoxin has been measured as an index of cytolysis. This newly developed CAT system is more sensitive than the traditional haemolysis method utilizing red blood cells or the lactate dehydrogenase assay for cytolysis. Series of chimaeric toxin molecules have been constructed by swapping the loops between highly hydrophilic neurotoxin and highly hydrophobic Cardiotoxin molecules from Naja sputatrix, which are known to exhibit structural similarity (three-finger conformation) but to have different functional properties. Comparison of the cytolytic activities of the recombinant chimaeric toxins demonstrated the possible involvement of all three loops of Cardiotoxin in its cytolytic potency. However, the first two loops of the protein appear to make the major contribution to its lytic activity. cDNAs encoding Cardiotoxin and the chimaeric toxins, when expressed in transfected cultured Chinese hamster ovary cells, resulted in cell lysis, indicating that these cDNAs can be developed as useful cytolytic agents.
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expression of Cardiotoxin 2 gene
FEBS Journal, 2001Co-Authors: Arunmozhiarasi Armugam, Kandiah JeyaseelanAbstract:This report is the first study of the regulation of expression of a toxin gene and it also demonstrates the novel finding that the Cardiotoxin (CTX)-2 gene from Naja sputatrix is expressed in the venom gland as well as in other tissues in the snake, such as liver, heart and muscle. The venom gland produces a 500-bp (spliced) CTX-2 mRNA as the final transcript. However, the liver produces two types of CTX-2 mRNA, of which the unspliced transcript (1 kb) is predominant; the 500 bp spliced transcript is the minor species. This differential expression of the CTX gene has been attributed to the usage of alternative promoter consisting of independent TATA boxes and corresponding transcription initiation sites. Among the several transcription factors that have been identified by a search of the TFIID database, the participation of two glucocorticoid elements in the expression of the CTX gene has been demonstrated by promoter deletion analysis. Putative binding sites for SP-1, C/EBP, CACCC-binding factor and at least two unknown binding factors have also been identified by DNase I footprinting of the promoter.
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structure and organization of the Cardiotoxin genes in naja naja sputatrix
FEBS Letters, 1998Co-Authors: Ramkumar Lachumanan, Arunmozhiarasi Armugam, Cheehong Tan, Kandiah JeyaseelanAbstract:Abstract We report the genomic structure, organization and the presence of multiple isoforms of the gene encoding Cardiotoxins (CTX) of Naja naja sputatrix . The Cardiotoxin gene consists of six CTX isoforms, each (2.2 kb) having three exons and two introns. Two possible transcription initiation sites as well as consensus TATA boxes and transcription factor binding motifs, AP-2, NFIL-6/C/EBP, NF-κB and PuF have been identified in the 5′-region of the gene. The CTX gene isoforms show nucleotide variations at specific segments in exon 2 and exon 3, which correspond to the functional domains in the three-finger loop structure of the Cardiotoxin molecule. The diverse functions of Cardiotoxins together with our findings suggest that the Cardiotoxin gene isoforms may have evolved under adaptive pressure through a positive Darwinian selection process.
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six isoforms of Cardiotoxin in malayan spitting cobra naja naja sputatrix venom cloning and characterization of cdnas
Biochimica et Biophysica Acta, 1998Co-Authors: Kandiah Jeyaseelan, Arunmozhiarasi Armugam, Ramkumar Lachumanan, C H Tan, Nget Hong TanAbstract:Cardiotoxins are the most abundant toxin components of cobra venom. Although many Cardiotoxins have been purified and characterized by amino acid sequencing and other pharmacological and biochemical studies, to date only five Cardiotoxin cDNAs from Taiwan cobra (Naja naja atra), three cDNAs from Chinese cobra (Naja atra) and two more of uncertain origin (either Chinese or Taiwan cobra) have been reported. In this paper we show the existence of four isoforms of Cardiotoxin by protein analysis and nine cDNA sequences encoding six isoforms of Cardiotoxins (CTX 1-3, 4a, 4b and 5) from N. n. sputatrix by cDNA cloning. This forms the first report on the cloning and characterization of several Cardiotoxin genes from a single species of a spitting cobra. The cDNAs encoding these isoforms, obtained by reverse transcription-polymerase chain reaction (RT-PCR), were subsequently expressed in Escherichia coli. The native and recombinant Cardiotoxins were first characterized by Western blotting and N-terminal protein sequencing. These proteins were also found to have different levels of cytolytic activity on cultured baby hamster kidney cells. Four of the isoforms (CTX 1, 2, 4 and 5) are unique to N. n. sputatrix, with CTX 2 being the most abundant species constituting about 50% of the total Cardiotoxins. The isoform CTX 3 (20% constitution) is highly homologous to the Cardiotoxins of N. n. atra and N. n. naja, indicating that it may be universally present in all Naja naja subspecies. Our studies suggest that the most hydrophilic isoform (CTX 5) could have evolved first followed by the hydrophobic isoforms (CTX 1, 2, 3 and 4). We also speculate that Asiatic cobras could be the modern descendants of the African and Egyptian counterparts.
Dingkwo Chang - One of the best experts on this subject based on the ideXlab platform.
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Cardiotoxin ii from taiwan cobra venom naja naja atra structure in solution and comparison among homologous Cardiotoxins
Journal of Biological Chemistry, 1994Co-Authors: R Bhaskaran, Chung Chih Huang, Yichun Tsai, Gurunathan Jayaraman, Dingkwo ChangAbstract:The three-dimensional structure in solution of Cardiotoxin II, a membrane toxin from the venom of Taiwan cobra, Naja naja atra, was determined using 1H nuclear magnetic resonance spectroscopy and molecular modeling based on the hybrid distance geometry/dynamic simulated annealing technique. A complete sequence-specific proton assignment was obtained, and the secondary structures of the protein were determined from information on nuclear Overhauser effect connectivities, coupling constants, and hydrogen exchange were confirmed using the main-chain-directed strategy. Twelve simulated annealing structures found to be within a single family were selected based on the condition of distance constraint violation less than 0.02 nm and the dihedral angle violation less than 4 degrees. The average atomic root mean square deviation between the selected structures and their geometric average are 0.079 nm for the backbone atoms and 0.137 nm for all heavy atoms; they are 0.044 nm and 0.117 nm, respectively, when considering the secondary structural residues only. The molecule adopts a compact structure consisting of three major loops emerging from a globular head. These loops contain five strands to form double- and a triple-stranded antiparallel beta sheets. Comparisons are made between this structure and those of its homologous Cardiotoxins in order to derive further information on their structural variations.
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Cardiotoxin iii from the taiwan cobra naja naja atra determination of structure in solution and comparison with short neurotoxins
Journal of Molecular Biology, 1994Co-Authors: R Bhaskaran, Chung Chih Huang, Dingkwo ChangAbstract:The structure in solution of Cardiotoxin III, a membrane toxin purified from the venom of the Taiwan cobra, Naja naja atra, is reported. Sequence-specific assignment of 1H-NMR lines was completed and the NMR data show the presence of a triple and a double-stranded antiparallel beta-sheet. Many NOE cross peaks identified in NOESY spectra were applied as distance constraints based on a hybrid distance geometry/dynamical simulated annealing technique; 20 structures were found within a single family. The average value of atomic RMS differences between the 20 structures and their geometric mean is 0.087 nm for the backbone atoms and 0.152 nm for all heavy atoms; they are 0.055 nm and 0.12 nm, respectively for the segments of secondary structure. In these selected structures the backbone of the polypeptide chain folds such that five strands emerge from a globular head. Three major loops link these strands to form a double and a triple-stranded antiparallel beta-sheet. Comparison of the structures of the toxin in solution with the X-ray crystal structure of its homologous protein, Cardiotoxin V4II from Naja mossambica mossambica, showed good agreement between the structures except at segments of the turns. As the functions of short neurotoxins and Cardiotoxins are distinct, despite their similar secondary structural patterns and tertiary folding, a comparative analysis has been carried out between Cardiotoxin III and short neurotoxins of known structures. We discuss their structural features in order to clarify relationships between their structure and function.
Arunmozhiarasi Armugam - One of the best experts on this subject based on the ideXlab platform.
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Cytotoxic potency of Cardiotoxin from Naja sputatrix: development of a new cytolytic assay.
Biochemical Journal, 2002Co-Authors: Arunmozhiarasi Armugam, Kandiah JeyaseelanAbstract:The possible involvement of specific regions/loops of Cardiotoxin from Naja sputatrix venom in mediating its cytolytic activity is evaluated using a new cytolytic assay. In this assay, the amount of chloramphenicol acetyltransferase (CAT) that is released upon lysis of the cellular membranes by the cytotoxin has been measured as an index of cytolysis. This newly developed CAT system is more sensitive than the traditional haemolysis method utilizing red blood cells or the lactate dehydrogenase assay for cytolysis. Series of chimaeric toxin molecules have been constructed by swapping the loops between highly hydrophilic neurotoxin and highly hydrophobic Cardiotoxin molecules from Naja sputatrix, which are known to exhibit structural similarity (three-finger conformation) but to have different functional properties. Comparison of the cytolytic activities of the recombinant chimaeric toxins demonstrated the possible involvement of all three loops of Cardiotoxin in its cytolytic potency. However, the first two loops of the protein appear to make the major contribution to its lytic activity. cDNAs encoding Cardiotoxin and the chimaeric toxins, when expressed in transfected cultured Chinese hamster ovary cells, resulted in cell lysis, indicating that these cDNAs can be developed as useful cytolytic agents.
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expression of Cardiotoxin 2 gene
FEBS Journal, 2001Co-Authors: Arunmozhiarasi Armugam, Kandiah JeyaseelanAbstract:This report is the first study of the regulation of expression of a toxin gene and it also demonstrates the novel finding that the Cardiotoxin (CTX)-2 gene from Naja sputatrix is expressed in the venom gland as well as in other tissues in the snake, such as liver, heart and muscle. The venom gland produces a 500-bp (spliced) CTX-2 mRNA as the final transcript. However, the liver produces two types of CTX-2 mRNA, of which the unspliced transcript (1 kb) is predominant; the 500 bp spliced transcript is the minor species. This differential expression of the CTX gene has been attributed to the usage of alternative promoter consisting of independent TATA boxes and corresponding transcription initiation sites. Among the several transcription factors that have been identified by a search of the TFIID database, the participation of two glucocorticoid elements in the expression of the CTX gene has been demonstrated by promoter deletion analysis. Putative binding sites for SP-1, C/EBP, CACCC-binding factor and at least two unknown binding factors have also been identified by DNase I footprinting of the promoter.
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structure and organization of the Cardiotoxin genes in naja naja sputatrix
FEBS Letters, 1998Co-Authors: Ramkumar Lachumanan, Arunmozhiarasi Armugam, Cheehong Tan, Kandiah JeyaseelanAbstract:Abstract We report the genomic structure, organization and the presence of multiple isoforms of the gene encoding Cardiotoxins (CTX) of Naja naja sputatrix . The Cardiotoxin gene consists of six CTX isoforms, each (2.2 kb) having three exons and two introns. Two possible transcription initiation sites as well as consensus TATA boxes and transcription factor binding motifs, AP-2, NFIL-6/C/EBP, NF-κB and PuF have been identified in the 5′-region of the gene. The CTX gene isoforms show nucleotide variations at specific segments in exon 2 and exon 3, which correspond to the functional domains in the three-finger loop structure of the Cardiotoxin molecule. The diverse functions of Cardiotoxins together with our findings suggest that the Cardiotoxin gene isoforms may have evolved under adaptive pressure through a positive Darwinian selection process.
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six isoforms of Cardiotoxin in malayan spitting cobra naja naja sputatrix venom cloning and characterization of cdnas
Biochimica et Biophysica Acta, 1998Co-Authors: Kandiah Jeyaseelan, Arunmozhiarasi Armugam, Ramkumar Lachumanan, C H Tan, Nget Hong TanAbstract:Cardiotoxins are the most abundant toxin components of cobra venom. Although many Cardiotoxins have been purified and characterized by amino acid sequencing and other pharmacological and biochemical studies, to date only five Cardiotoxin cDNAs from Taiwan cobra (Naja naja atra), three cDNAs from Chinese cobra (Naja atra) and two more of uncertain origin (either Chinese or Taiwan cobra) have been reported. In this paper we show the existence of four isoforms of Cardiotoxin by protein analysis and nine cDNA sequences encoding six isoforms of Cardiotoxins (CTX 1-3, 4a, 4b and 5) from N. n. sputatrix by cDNA cloning. This forms the first report on the cloning and characterization of several Cardiotoxin genes from a single species of a spitting cobra. The cDNAs encoding these isoforms, obtained by reverse transcription-polymerase chain reaction (RT-PCR), were subsequently expressed in Escherichia coli. The native and recombinant Cardiotoxins were first characterized by Western blotting and N-terminal protein sequencing. These proteins were also found to have different levels of cytolytic activity on cultured baby hamster kidney cells. Four of the isoforms (CTX 1, 2, 4 and 5) are unique to N. n. sputatrix, with CTX 2 being the most abundant species constituting about 50% of the total Cardiotoxins. The isoform CTX 3 (20% constitution) is highly homologous to the Cardiotoxins of N. n. atra and N. n. naja, indicating that it may be universally present in all Naja naja subspecies. Our studies suggest that the most hydrophilic isoform (CTX 5) could have evolved first followed by the hydrophobic isoforms (CTX 1, 2, 3 and 4). We also speculate that Asiatic cobras could be the modern descendants of the African and Egyptian counterparts.
R Bhaskaran - One of the best experts on this subject based on the ideXlab platform.
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Cardiotoxin ii from taiwan cobra venom naja naja atra structure in solution and comparison among homologous Cardiotoxins
Journal of Biological Chemistry, 1994Co-Authors: R Bhaskaran, Chung Chih Huang, Yichun Tsai, Gurunathan Jayaraman, Dingkwo ChangAbstract:The three-dimensional structure in solution of Cardiotoxin II, a membrane toxin from the venom of Taiwan cobra, Naja naja atra, was determined using 1H nuclear magnetic resonance spectroscopy and molecular modeling based on the hybrid distance geometry/dynamic simulated annealing technique. A complete sequence-specific proton assignment was obtained, and the secondary structures of the protein were determined from information on nuclear Overhauser effect connectivities, coupling constants, and hydrogen exchange were confirmed using the main-chain-directed strategy. Twelve simulated annealing structures found to be within a single family were selected based on the condition of distance constraint violation less than 0.02 nm and the dihedral angle violation less than 4 degrees. The average atomic root mean square deviation between the selected structures and their geometric average are 0.079 nm for the backbone atoms and 0.137 nm for all heavy atoms; they are 0.044 nm and 0.117 nm, respectively, when considering the secondary structural residues only. The molecule adopts a compact structure consisting of three major loops emerging from a globular head. These loops contain five strands to form double- and a triple-stranded antiparallel beta sheets. Comparisons are made between this structure and those of its homologous Cardiotoxins in order to derive further information on their structural variations.
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Cardiotoxin iii from the taiwan cobra naja naja atra determination of structure in solution and comparison with short neurotoxins
Journal of Molecular Biology, 1994Co-Authors: R Bhaskaran, Chung Chih Huang, Dingkwo ChangAbstract:The structure in solution of Cardiotoxin III, a membrane toxin purified from the venom of the Taiwan cobra, Naja naja atra, is reported. Sequence-specific assignment of 1H-NMR lines was completed and the NMR data show the presence of a triple and a double-stranded antiparallel beta-sheet. Many NOE cross peaks identified in NOESY spectra were applied as distance constraints based on a hybrid distance geometry/dynamical simulated annealing technique; 20 structures were found within a single family. The average value of atomic RMS differences between the 20 structures and their geometric mean is 0.087 nm for the backbone atoms and 0.152 nm for all heavy atoms; they are 0.055 nm and 0.12 nm, respectively for the segments of secondary structure. In these selected structures the backbone of the polypeptide chain folds such that five strands emerge from a globular head. Three major loops link these strands to form a double and a triple-stranded antiparallel beta-sheet. Comparison of the structures of the toxin in solution with the X-ray crystal structure of its homologous protein, Cardiotoxin V4II from Naja mossambica mossambica, showed good agreement between the structures except at segments of the turns. As the functions of short neurotoxins and Cardiotoxins are distinct, despite their similar secondary structural patterns and tertiary folding, a comparative analysis has been carried out between Cardiotoxin III and short neurotoxins of known structures. We discuss their structural features in order to clarify relationships between their structure and function.